MBL is a protein of the collectin family and characterised by an oligomeric structure of subunits. Each subunit consists of 3 identical polypeptides each consisting of a calcium-dependent, C-type carbohydrate-recognition domain (CRD), attached to a collagenous rod.
The number of subunits in an MBL molecule is varying (Lipscombe R J, et al, 1995), but it has been suggested that the biologically active polypeptide is an oligomer consisting of more than three subunits. Naturally occurring MBL in plasma comprises mainly oligomers of more than three subunits, and in addition denatured and structurally impaired protein forms leading to bands on for example SDS gels between the dominating MBL bands corresponding to the higher oligomers.
Recombinantly produced MBL reveals an oligomer variation similar to plasma-derived MBL (Vorup-Jensen T et al., 2001). However, usually recombinantly produced MBL has a higher content of low-mass forms than do plasma derived MBL. Low-mass forms of MBL include for example single polypeptide chains, single subunits, and dimeric subunits.
MBL is structurally related to the C1q subcomponent of component C1 of complement, and it appears that MBL activates the complement system via associated serine protease termed MASPs (Matsushita, M., 1992) or p100 (Ji, Y -H. et al., 1993), which are similar to the C1r and C1s components of the classical pathway. The MBL complement activation pathway is called the MBLectin pathway. According to the mechanism postulated for this pathway, MBL binds to specific carbohydrate structures found on the surface of a range of microorganisms including bacteria, yeast, parasitic protozoa and viruses (Turner, M. W, 1996), and its antimicrobial activity results from activation of the terminal, lytic complement pathway components (Kawasaki, N et al., 1989) or promoting phagocytosis (Kuhiman, M et al., 1989).
Reportedly, the level of MBL in plasma may be genetically determined (Sumiya, M et al., 1991, Lipscombe, R. J. et al., 1992, Madsen H. O. et al. 1994). MBL deficiency is associated with susceptibility to frequent infections with a variety of microorganisms in childhood (Super, M et al., 1989, Garred, P. et al., 1995), and possibly, in adults (Garred, P. et al., 1995, Summerfield, J. A. et al. 1995). Recent information associates MBL deficiency with HIV infection and with more rapid death following development of AIDS (Nielsen, S. L et al., 1995, Garred, P et al., 1997). MBL binds to the a galactosyl form of IgG (G0), which is found at elevated concentrations in rheumatoid arthritis patients, and then activates complement (Malhotra, R et al., 1995). MBL deficiency is also associated with a predisposition to recurrent spontaneous abortions (Kilpatrick, D. C et al., 1995), and also to development of systemic lupus erythrematosus (Davies, E. J et al., 1995).
MASP-1 (MBL-associated serine protease 1) is a serine protease similar in structure to C1r and C1s of the complement pathway although it has a histidine loop structure of the type found in trypsin and trypsin-like serine proteases. MASP-1 has been found to be involved in complement activation by MBL. A cDNA clone encoding MASP-1 has been reported that encodes a putative leader peptide of 19 amino acids followed by 680 amino acid residues predicted to form the mature peptide.
MASP-2 (MBL-associated serine protease 2) (Thiel, S et al., 1997) is a serine protease similar in structure to C1r and C1s of the complement pathway. Like these, and contrary to MASP-1, it has no histidine loop structure of the type found in trypsin and trypsin-like serine proteases. MASP-2 has been found to be involved in complement activation by MBL. MASP-3 shows some homology with MASP-1 and MASP-2 and the two C1q-associated serine proteases, C1r and C1s.
MBL purified from serum has previously been used in a pharmaceutical formulation furthermore containing human serum albumin to treat two MBL deficient individuals and said MBL treatment resulted in significant reduction of infections (Valdimarson et al., 1998). In addition, various other pharmaceutical MBL formulations have been described in the prior art:
Valdimarsson et al. describes infusion of a solution containing 200 μg/ml MBL in 0.15 M NaCl and 1% w/v (=10 mg/ml) human serum albumin.
U.S. Pat. No. 5,270,199 describes an MBL formulation comprising 5 to 100 μg/ml MBL and/or MBL fragment.
WO 99/64453 describes various MBL formulations comprising MBL and a stabilising agent. As preferred example is mentioned a formulation comprising 300 to 400 μg/ml MBL in PBS with 0.5% w/v albumin (=5 mg/ml).
However, these MBL formulations comprise either low amounts of MBL and/or high concentration of other proteins.